Support for development of the present invention was received from the United States Department of Health, Education and Welfare.
The present invention relates generally to the field of multidimensional electrophoretic analysis and, more particularly, but not by way of limitation, to an apparatus for conducting bidimensional or orthogonal electrophoretic analyses of molecular species in an open flat bed gel.
Molecules or particles having net electrical charges may be distinguished by observing their migrations in suitable media subjected to the application of electrical fields. Such migrations are dependent upon the composition of the media, the nature of the electrical fields and characteristics of the molecules or particles themselves, such as net surface electrical charge, size and shape.
Complex mixtures of, for example, proteins, peptides or nucleic acids have long been analyzed by electrophoresis in various media. Among the more popular media for these analyses have been polyacrylamide and agarose gels containing an electrically conductive pH buffer.
Both columnar and slab electrophoretic apparati have been utilized for gel electrophoresis of various molecules. Unidimensional gel electrophoretic separation procedures are well known. Numerous approaches have been made toward establishing reproducible procedures for bidimensional gel electrophoretic separations of molecules. Most popularly, these approaches have involved electrophoretic separation in a vertical columnar gel, particularly polyacrylamide, followed by extraction of the gel column. The extracted columnar gel, bearing molecules separated along its length, has been then embedded in a gel slab having different characteristics such as pH, porosity or denaturant content, for example. An electric field generally perpendicular to the embedded gel was then applied to cause a molecular migration orthogonal to the original electric field applied to the columnar gel. This approach to bidimensional electrophoretic analysis is subject to several difficulties. First, the physical manipulations required to extract the columnar gel from its retaining column often result in gel deformation or breakage. For an intact columnar gel to be extractable at all from a column, the gel medium must have sufficient tensile strength to withstand handling. Experience has shown desirable gel media such as, for example, 0.3 to 0.4% agarose gels, cannot be extracted from a gel column without breakage. Because an extracted columnar gel, particularly a gel with low gel content, is difficult to embed in a precise linear orientation, further irreproducibility of migrations in the second electrophoretic dimension may arise.
In U.S. Pat. No. 4,385,974, issued to Shevitz a slab gel bidimensional electrophoresis apparatus is described. This apparatus included two pairs of opposing tanks, electrode means in each tank and means for supporting a gel slab defined by the space between a pair of parallel gel plates, a pair of barrier webs and two gels.